L J (é’flfl\\\\ L ‘- . “130 W is] v.“" OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Phce in book return to remove charge from circulation records THE EFFECTS OF CALCIUM ON THE RED BLOOD CELL IN A HYPOTONIC ENVIRONMENT BY Robert Henry Williams A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1979 ABSTRACT THE EFFECTS OF CALCIUM ON THE RED BLOOD CELL IN A HYPOTONIC ENVIRONMENT BY Robert Henry Williams Red blood cell sizing and cation studies were determined at 25°C, in isotonic and hypotonic sodium chloride solutions, pH adjusted to 7.40, in the presence and absence of calcium. Sizing studies were determined employing an improved electronic particle counting method, using glutaraldehyde fixed red cells, which permitted the precise construction of swelling curves. The swelling curves of calcium- treated cells were shifted towards the lower osmolalities compared to controls. Calcium—exposed cells also demonstrated a greater loss of potassium with a less than equal gain of sodium, which was analo- gous to the Gardos phenomenon, but different in that 1) during the period of osmotic stress, cells were not ATP depleted and 2) there was a calcium-promoted effect on the magnitude of sodium influx. The results suggest that calcium, to which the red cell is basically impermeable, is able to penetrate the membrane and provide a mechanism for increasing the osmotic resistance of the cell. TO KENNETH R. WILLIAMS: my son ii ACKNOWLEDGEMENTS I wish to acknowledge the assistance of the members of my graduate advisory committee: Dr. Anthony J. Bowdler of the Depart- ment of Medicine; Mrs. Martha T. Thomas and Dr. George A. Padgett of the Department of Pathology; Dr. Robert W. Leader, Chairman of the Department of Pathology; and Cynthia M. D'Antonio. iii TABLE OF CONTENTS Page LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . l Osmotic Fragility. . . . . . . . . . . . . . . . . . . . 1 Introduction. . . . . . . . . . . . . . . . . . . l Ponder's Equation: A Mathematical Expression of Red Blood Cell Osmometric Behavior. . . . . 2 Explanations for the Physical Basis of Ponder's "R" . . . . . . . . . . . . . . . . . 8 Osmotic Lysis: Electrolytes and Non-Electrolytes in the Suspending Medium . . . . . . . . . . . l4 Theories to Explain Red Blood Cell Resistance to Osmotic Lysis . . . . . . . . . . . . . . . 16 Calcium - Effects on Red Blood Cell Function . . . . . . 22 Introduction. . . . . . . . . . . . . . . . . . . 22 Effects on Red Cell ATPases . . . . . . . . . . . 22 Effects on Membrane Structure and Function. . . . 27 METHODS AND MATERIALS . . . . . . . . . . . . . . . . . . . . . 30 Introduction: Electrical Particle Sizing and Glutaraldehyde Fixation . . . . . . . . . . . . . . . 30 Determination of Appropriate Glutaraldehyde Concen- tration for Particle Sizing . . . . . . . . . . . . . 34 Red Cell Sizing in Hypotonic Solutions of Sodium Chloride Containing Different Concentrations of Calcium. . . . . . . . . . . . . . . . . . . . . . 36 The Effect of Cell Shape on Red Blood Cell Sizing. . . . 42 Determination of the Cation Content of Osmotically Stressed Cells at Various Calcium Concentrations. . . 46 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 The Effect of Red Cell Shape on Particle Sizing. . . . . 49 Particle Sizing and Red Blood Cell Swelling. . . . . . . 49 Calcium Effects on the Relative Swelling of Red Blood Cells . . . . . . . . . . . . . . . . . . . . . 49 The Effect of Calcium on the Cation Content of Red Blood Cells . . . . . . . . . . . . . . . . . . . . . 59 SUMMARY AND CONCLUSION. . . . . . . . . . . . . . . . . . . . . 71 LIST OF REFERENCES. . . . . . . . . . . . . . . . . . . . . . . 74 VITA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 iv Table 10 ll 12 13 LIST OF TABLES Comparison of mean channel number as a function of glutaraldehyde concentration and 300 mOsm . Comparisons of Comparisons Comparisons Comparisons Comparisons Comparisons Comparisons Comparisons Comparisons Comparisons Comparisons Comparisons of of of of of of of of of of of red cell red cell red cell red cell red cell red cell red cell red cell red cell red cell red cell red cell fixation time at relative swelling at 300 mOsm. . relative swelling at 160 mOsm. . relative swelling at 120 mOsm. sodium content at 300 mOsm . . . potassium content at 300 mOsm. . total cation content at 300 mOsm sodium content at 160 mOsm . . . potassium content at 160 mOsm. . total cation content at 160 mOsm sodium content at 120 mOsm . . . potassium content at 120 mOsm. . total cation content at 120 mOsm Page 35 56 57 58 62 63 64 65 66 67 68 69 7O Figure LIST OF FIGURES The family of Ponder curves. . . . . . . . . . . . . . The visco-elastic properties of the red blood cell membrane . . . . . . . . . . . . . . . . . . . . . . . Schematic representation of chloride-hydroxyl ion exchange, coupled to the buffering action of hemoglobin . . . . . . . . . . . . . . . . . . . . . . The effect of glutaraldehyde fixation on the sizing of red blood cells . . . . . . . . . . . . . . . . . . Spinning technique for removing ghosts . . . . . . . . The effect of particle shape on particle sizing. . . . The relative change in red blood cell volume using fixed red blood cells and an electronic particle Counter 0 C O O O O O O O O O O O O O O I . O O O O O O The effects of calcium on the relative swelling of red blood cells. . . . . . . . . . . . . . . . . . . . The effect of calcium on red blood cell cation content vi Page 13 18 33 4O 51 53 55 61 LITERATURE REVIEW Osmotic Fragility Introduction Clinical interpretation of measurements of osmotic fragility depends on the concept that erythrocyte fragility is determined by the relationship of cell surface area to cell volume. The limiting case occurs when the maximum volume permitted by the surface area has been achieved and the shape has become spherical. At this point, further increase in volume is impossible, and if the membrane resistance to stress is minimal, lysis of the cell must occur. The classical theory of this process requires that an observed increase in fragility is interpretable as resulting from a decrease in the surface area/ volume ratio, by which the cells attain a more spherical form. A decrease in fragility is therefore regarded as due either to an increase in surface area or to a decrease in osmotically active cell content. In general, the erythrocyte has been considered to be a balloon— like structure encompassed by a semipermeable membrane which does not resist the changes in volume brought about by osmotic forces, and is unaffected in its properties by the hypotonic or hypertonic environ- ment. Any volume change which occurs in a hypotonic or hypertonic medium is the result of water transfer only. With these assumptions, the red blood cell could be anticipated to behave as a perfect 2 osmometer (83). However, it is apparent experimentally that the red blood cell deviates from the expectations of perfect osmometric behavior, and it is therefore anticipated that an understanding of the deviations would provide further insight into the physicochemical system of the red blood cell. These deviations are in part explicable in terms of the anomalous osmotic effects of concentrated protein within the cell. Furthermore, the erythrocyte membrane is a highly selective barrier with complex properties which can be a further source of deviation from simple theoretical prediction. Ponder's Equation: A Mathematical Expression of Red Blood Cell Osmometric Behavior If the erythrocyte behaves as a perfect osmometer, with its interior initially in osmotic equilibrium with the surrounding plasma, and if any volume change which occurs in a hypotonic or hypertonic medium is the result of water transfer alone, then the new volume (V) which the cell attains in a very large volume of a medium of tonicity (T) will be: 1 V = W ( T.- l ) + 100 This is one form of the "van't Hoff-Mariotte law" for ideal osmotic swelling. The initial volume is denoted by 100, W is the percentage of the cell volume made up of water, and the tonicity (T) is defined as the ratio of the depression of the freezing point (osmolality) of the suspending medium to that of plasma. For example, if the tonicity of a hypotonic sodium chloride solution is 0.5 (equivalent to 150 mOsm divided by 300 mOsm for isotonicity of a sodium chloride medium compared to plasma), the new volume (V) is equal to 170, assuming the percentage of cell water (W) is 70. However, most experimental results 3 have departed from this relationship. Observed volumes of red blood cells in a hypotonic environment are frequently less than expected (83); conversely, in hypertonic media the volumes are larger (llO). Ponder noted that deviations from the expected relationship vary with the composition of the hypotonic medium in which red blood cells are suspended. For example, volume changes in hypotonic sodium chloride solutions are less than those in hypotonic plasma at the same measured osmolalities. Another source of variation arises from the specific anticoagulant employed (79). Ponder further observed addi- SrCl and tional deviations in hypotonic solutions of BaCl 2, 2 2, MgCl CaCl2 (81). To allow mathematically for these deviations from theory, Ponder introduced the empirical factor R: l V=RW(¥-l)+100 This expression is known as Ponder's equation. The smaller the value of R, the less is the change of volume observed when the tonicity is changed. In other words, the cell behaves as a less perfect osmometer. Conversely, the closer R is to 1.0, the more closely the cell resembles a perfect osmometric system. R values for defibrinated and oxalated blood suspended in hypotonic plasma are approximately 0.8-0.9 and 0.5-0.7, respectively (83). Generally, R values in hypotonic plasma are higher than those observed in hypotonic sodium chloride (83). In order to assess the value of R accurately in a hypotonic medium, swelling curves are constructed by measurements of volume under hypotonic conditions and expressed as a fraction of the isotonic volume. The fractional change is plotted on the ordinate versus tonicity or osmolality on the abscissa (Figure l). The graph is curvilinear, when illustrated in this way. R can be calculated for Figure l. The family of Ponder curves. Each curve is repre- sented by a different value of RW for Ponder's equation. V = RW ( %'- l ) + 100 Curve 5W a’ 90 a 70 b 56 c 42 d 28 e 14 f 7 The initial volume is denoted by 100, the tonicity (T) is defined as the depression of the freezing point (osmolality) of the suspend- ing medium to that of plasma, W is the percentage of the cell volume made up of water, and the new volume which the cell attains is V and is given in percent. Dividing the new volume (V) by 100 gives the fractional increase in cell volume which is represented in this figure. ISOTONIC VOLUME OF 1.800T I 1.700‘ 1.600“ 1300-- 300 -P b I 1.0 0.8 250 100 OSMOLALITY (mOSM / kg) 1 l r I 0.6 0.4 Tomcrrv (T) FIGURE 1 «r oi 6 each curve, provided that the percentage of cell water at isotonicity is known. Otherwise, the constant for each curve can be symbolized by RW, which represents the apparent osmotically active water content. When the red blood cell behaves as a perfect osmometer, R equals 1.0 and all volume changes are due only to water transfer. Therefore, any change in R suggests the occurrence of factors in the cell or suspending medium which influence the diffusion processes of water, such as variable osmotic coefficients of the cell contents. The family of curves illustrated in Figure l are known as the Ponder curves with each curve representing a different value of RW. If the water content of human red blood cells is approximately 70% (82,40), then dividing RW by seventy gives the corresponding R value for each curve. For example, curve "a" represents a population of red blood cells which behaves as a perfect osmometer if the cell water content is 70%. Curve "a’" represents the same population of cells, except that the active cell water content appears to be 90%. The other curves describe red cell populations which progressively depart from behavior as perfect osmometers. Their RW value is less than 70; therefore, R is less than 1.0. It is not, of course, necessary that a swelling curve should superimpose on one of these curves. It may lie between two curves depending upon the value of RW for the tested red cell population; however, this permits estimation of the RW value which can be confirmed by empirical curve fitting. Besides giving information on the osmometric behavior of red blood cells, swelling curves can be used to calculate the mean critical hemolytic volume (Vh), which is the maximum volume a population of cells can attain prior to lysis. 7 There are three factors which affect the experimental determina- tion of Ponder's R: the tonicity (T), the percentage of cell water (W), and the measured cell volume (V). Technically, tonicity can be accurately determined by depression of freezing point. Cell water determinations can be estimated by a time consuming procedure. How- ever, for many purposes it is sufficient to make the assumption that normal red blood cells have a mean water content of approximately seventy percent (40,82). The maximum error of this assumption is unlikely to be greater than approximately seven percent. However, this error is increased if it is applied to abnormal cells. For example, sickle cells and desiccocytes have a decreased water content and do not swell to the same degree as normal red blood cells when suspended in a hypotonic medium (83,20). In estimating R it is critically important to have an accurate means of measuring cell volume. If the method of measuring red blood cell volume bears a source of uncertainty, then estimates of R will be inaccurate. Previous techniques for measuring red blood cell volume have had limitations, especially at hemolytic tonicities (78). For example, with the colorimetric method, a colored substance is added to a volume of blood and to a volume of plasma about equal to the volume of plasma which the blood contains. The cells are then centrifuged down from the sample of blood and the color of the super- natant plasma is compared with that of the plasma which contains the coloring substance in known concentration. Hemoglobin from the same animal or person is usually employed as the colored substance because it neither penetrates the cell nor is it absorbed onto its surface. If hemoglobin is liberated by the red blood cells in the system, the whole procedure becomes invalid. The hematocrit method depends upon 8 being able to measure the height of the column of packed cells. If cells have hemolyzed, a correction must be employed to determine the true packed cell volume of the residual cells. Regardless of hemolysis, packing factors associated with different cell shapes and volumes will affect hematocrit measurements. Variable shape and volume are also factors in precluding the use of refractometer, conductivity, and viscosity methods (83). Korpman et al. have also noted the limitation of diffraction measurements as well (53). The use of electronic particle counters is feasible provided that the significance of the output is critically evaluated. Red blood cells, which are not pre- served with fixatives, give inaccurate distribution curves due to: l) variability in instrument response due to deformability of the cells as they pass through the orifice (13,39), 2) changes in conduc- tivity due to differences in ionic strength between solutions in the counting apparatus and red cell suspensions of varying tonicities (67), 3) artifacts produced by red cell ghosts (hemolyzed red blood cells) (113), and 4) variable effects produced by flow (aspiration) rates resulting in incomplete signal development (50,104). Therefore, it becomes apparent that the calculation of Ponder's R is influenced by several parameters and its interpretation can only be as accurate as the methods which are used to determine it. Explanations for the Physical Basis of Ponder's "R" There have been several explanations proposed with respect to the physical basis of R (76); of these, the following have been the most discussed: 1) Leakage of ions from red blood cells into the medium 2) Binding of water of solvation to red cell proteins 9 3) Dependence of the osmotic coefficients and electrical charge of hemoglobin on the intracellular protein concentration 4) Inherent resistance of the red blood cell to deforma- tion when swelling. 1) Any leakage of ions from the cell into the suspending medium reduces the amount of the osmotically active substances, thereby reducing the volume increase due to the influx of water into the cell. One source of this phenomenon is the prelytic loss of K+ (80,98,24). However, the K+ losses which have been experimentally determined do not account for the small values of R which have been observed (80,43,76). Furthermore, there are hypotonic suspensions that are close to isotonicity which show no prelytic K+ leak (69,24,26). 2) If part of the cell water is bound to protein, then there is a reduced proportion in the amount of osmotically active water. This concept, simply stated, implies that the measurable cell water is not an adequate measure of the osmotically available cell water, which is therefore less than W. If the percentage of cell water taking part in volume changes (W) is apparently decreased, the amount of volume increase will be less than predicted. The hypothesis has lost popu- larity with several investigators (83,82,76,26). 3) It is known that the osmotic coefficient of many proteins, including hemoglobin, rises sharply with concentration. If erythro- cytes are transferred from an isotonic to a hypotonic medium, the concentration of hemoglobin decreases due to cellular swelling. Consequently, there is a decrease in the osmotic coefficient of hemo- globin. This decrease in protein contribution to the total osmolality of the cell makes the swelling of the cell proportionately less as 10 the volume progressively increases (76,24,26,109). Conversely, in hypertonic solutions, as the hemoglobin concentration increases, there is a concomitant increase in its osmotic coefficient. There- fore, the outflow of water from the cell is less than expected and the cell shrinks less. The properties of hemoglobin affect cell volume by a second mechanism: Hladky and Rink (43) propose that the net charge on hemoglobin approaches zero as its concentration increases. At a pH above its isoelectric point, hemoglobin carries a net nega- tive charge. If a red blood cell is suspended in a hypertonic medium, the concentration of hemoglobin increases as the cell becomes smaller. Consequently, the charge on the protein becomes less. To maintain electroneutrality, chloride diffuses into the cell, thus increasing the solute content of the cell. Water follows passively, thus preventing the cell from shrinking fully to its expected size. Conversely, if the cell is placed in a hypotonic medium, the net nega— tive charge on the hemoglobin increases. Chloride then diffuses out of the cell with a concomitant efflux of water. Therefore, the magni- tude of cell swelling is diminished. 4) The red blood cell has a membrane which appears to be a semisolid possessing visco-elastic properties (33). Most of these properties have been attributed to the spectrin-actin complex (64) because the membrane lipids are fluid at 37°C (57). The membrane is capable of large elastic extensions with complete recovery unless the extension exceeds its yield point (44,32,57.18). These elastic responses occur in the form of deformations produced by bending or distorting the cell membrane (57,44). Apparently, the red cell has a limited capacity for elastic change in the plane of its membrane. Therefore, elastic change or deformation results at most in a very ll limited increase in surface area. However, if the red blood cell membrane is subjected to forces which increase its extensibility beyond its yield point, plastic change may occur resulting in irre- versible deformation (Figure 2). Classical theory implies that the maximum surface area attained by a prelytic sphere is equal to that of the discocyte from which it originated (83,40,82). In the absence of membrane expansion, the cell produces a shape change. The visco-elastic properties of the membrane permit cell deformability with respect to shape, but limit the tolerance of the membrane to the increased tensions which may result in plastic change (83,49). Other investigators believe that the membrane is capable of limited expansion and have suggested that the membrane can expand its surface area 7-20% (86,51). Recent observations by Evans et a1. lead to the conclusion that the amount of membrane resistance produced by osmotic tension is high in the plane of the membrane. Their findings indicate that the red blood cell membrane can expand its surface area by only 3% when osmotically swollen (34). Waugh and Evans have recently shown that the extensi- bility of the red blood cell membrane is temperature dependent. Surface area changes were compared to those at 25°C. At higher temperatures the membrane can increase its elasticity and expand its surface area. Conversely, at lower temperatures the elasticity of the membrane is decreased (105). However, it should be pointed out that the suspending medium can influence the membrane in such a way as to change its extensibility (51,52,69). Therefore, the effect of the inherent membrane resistance to swelling in a hypotonic medium may be dependent upon several factors (temperature, suspending medium) which may directly affect the membrane extensibility and therefore R. 12 Figure 2. The visco-elastic properties of the red blood cell membrane. The membrane is capable of large elastic extensions which occur in the form of a shape change. As long as the extension of the membrane produces no appreciable change in surface area, elastic deformation occurs and the red blood cell is able to reassume a biconcave disc shape. However, if the extension results in a change in the surface area, the cell membrane is unable to recover its elasticity and plastic (permanent) deformation occurs. 13 RED BLOOD CELL A) B) <__ ; C) I l i D) <_: I I FIGURE 2 ___J ENLARGED SQUARE ELEMENT OF MEMBRANE SURFACE DEFORMATION INTO RECTANGLE SURFACE AREA CONSTANT ELASTIC DEFORMATION DEFOR NATION INTO RHOMBUS SURFACE AREA CONSTANT ELASTIC DEFORMATION SURFACE AREA DILATATION PLASTIC DEFORMATION 14 Also, the question remains as to what effect submembrane struc- ture (stroma) may have on shape change and membrane modification which may put limitations on swelling or shrinking and the hemolytic process. The high concentration of hemoglobin in the red blood cell interior and the occurrence of stromatin, which can form gels at very low concentrations, indicate that the liberation of hemoglobin in the red blood cell could be greatly influenced by strong intermolecular forces (41). This simply implies that swelling and hemoglobin release may be a complicated process involving the loosening of intermolecular bonds with a rearrangement of the underlying substructure. Osmotic Lysis: Electrolytes and Non- Electrolytes in the Suspending Medium It has been known for some time that the osmotic fragility of red cells is dependent upon the composition of the suspending medium. The lysis of red blood cells varies in degree depending upon the presence of electrolytes and/or non-electrolytes (25,56). Electro- lytes can affect osmotic fragilities in a different manner depending upon the major cation present. Ponder has observed that the osmotic fragilities of red blood cells suspended in hypotonic solutions of the chlorides of the alkaline earths are different. Osmotic fragility is apparently decreased in the order NaCl > BaCl > MgCl > SrCl2 (81). 2 2 Mann and Collier have noted that buffered hypotonic NaCl solutions containing calcium decrease the amount of hemolysis compared to NaCl solutions which contained no calcium (66). Lake et al. found that loading erythrocytes with Ca2+, using an ionophore, decreases the osmotic fragility due to the efflux of KI from the red blood cells. The decrease in osmotically active substances reduces the amount of swelling of the red blood cells and therefore retards hemolysis (58). 15 Bowdler and Chan have demonstrated that red blood cell lysis in hypo- tonic electrolyte solutions is divided into two phases: an early rapid phase and a secondary smaller phase which has a longer half- time (8). The magnitude of the second phase is dependent on the dominant external cation, becoming progressively greater through the series: Mg2+ < Na+ < Li+ < K+ < Rb+. The slow phase is interpreted as arising from increased passive cation permeability in cells swollen to a volume close to that critical for hemolysis, with water influx secondary to the unopposed colloid osmotic pressure of intra- cellular protein. This slow phase of hemolysis is inhibited by the addition of 20 mM sucrose to the hypotonic medium. Wessels and Veerkamp also have indicated that slow phase hemolysis can be modi- fied or abolished by non-penetrating non-electrolytes such as sucrose (108). Furthermore, they have shown that buffers can affect the degree of lysis (109). They hypothesize that in buffered sucrose solutions an exchange of intracellular chloride against extracellular hydroxyl ions will increase the intracellular pH resulting in a con- comitant liberation of protons from hemoglobin. The extracellular pH will decrease. These processes cause a decrease of the intra- cellular osmotic activity, since the inorganic anion concentration is lowered. Conversely, in buffered sodium chloride solutions there will be an influx of chloride ions, an efflux of hydroxyl ions and a protonation of hemoglobin, which together results in an increase of the intracellular osmotic value. Therefore, the initial composi- tion, the pH and the buffering capacity of the extracellular solution will affect the following parameters: (a) the direction and extent of chloride-hydroxyl exchange, (b) the intracellular and extracellular pH, (c) the net charge of hemoglobin, and (d) the mean cellular volume l6 and the degree of swelling and, therefore, the degree of lysis (Figure 3). Meryman and others have demonstrated that red blood cells suspended in hypotonic solutions of a non-penetrating non-electrolyte such as sucrose cause a sharp increase in K+ leak which introduces a sub- stantial error into measurements involving hypotonic hemolysis (27,56,68,69). This phenomenon reduces the amount of intracellular osmotically active solute, causing hemolysis to occur at a lower osmolality than would be expected for a cell with a normal solute content. Apparently, the loss of K+ from red blood cells is related to the ionic strength of the solution and a minimum amount of elec— trolyte is needed to maintain membrane integrity (25,69). Davies et al. have reported that hemoglobin loss in hypotonic solutions of other non-penetrating non-electrolytes such as polymers (albumin, dextran, polyethylene glycol, and ficoll) is less than that in sodium chloride, although hemolysis begins to be seen at about the same osmolality (23). Theories to Explain Red Blood Cell Resistance to Osmotic Lysis When a population of red blood cells is suspended in hypotonic solutions that produce 100% hemolysis, only fast phase hemolysis occurs; that is, hemolysis is produced only by a rapid entry of water. However, if a population of red blood cells is suspended in a hypotonic solu- tion which induces partial hemolysis, two phases develop: a fast phase followed by a slow phase of longer duration (17,8). As noted earlier, the slow phase is believed to arise from cation permeability in cells swollen close to their critical hemolytic volume with water influx secondary to the unopposed colloid osmotic pressure of 17 Figure 3. Schematic representation of chloride-hydroxyl ion exchange, coupled to the buffering action of hemoglobin. X repre- sents an imidazole or amino group and Y a carboxylic group. Reprinted from (109). 18 FIGURE 3 _hnflnmd_mcm_ W m mm mm m Cl"——9CI‘ +HX 4. X" \ ouz— 0H- H20 -—+H,O YHJ c|'—————.cr’ ”V H20 ——§H20 x OH?—— OH” “I'Hx l9 intracellular protein. This phase occurs whether cells are subjected to rapid or gradual hemolysis. Rapid hemolysis and gradual hemolysis refer to the rate at which the hypotonic solution is added. In rapid hemolysis, cells are added directly to the hypotonic medium, whereas in gradual hemolysis cells are added to an isotonic medium, the ionic strength of which is gradually diminished by dialysis or infusion of a hypotonic solution. In either case, a population of intact cells remains. To explain the apparent osmotic resistance of residual cells subjected to slow phase hemolysis, two major theories are proposed: 1) Katchalsky's membrane expansion model 2) Seeman's K+ leak model. These are briefly discussed. 1) Katchalsky's model is based on the hypothesis that when the influx of water is slow, as in gradual or slow phase hemolysis, the expansion of the cell membrane follows the rate of swelling and no stresses develop in the membrane until a critical volume is reached. However, when the water influx is high, as in fast hemolysis, additional stresses develop in the membrane. This leads to ultimate rupture of the membrane and subsequent hemolysis (51). This theory implies that the membrane is capable of expanding provided that the rate of change of stress is gradual. Furthermore, it infers that the surface area can change depending upon the rate of stress. Consequently, the critical hemolytic volume should be higher than the critical hemolytic volume obtained in fast hemolysis. Sodium-potassium leak associated with colloidal osmotic-lysis occurs near the critical hemolytic volume and is equimolar. The cell hemolyzes at a lower tonicity during gradual hemolysis because the membrane is capable of limited membrane 20 expansion. Therefore, Katchalsky suggests that the membrane is visco- elastic and is capable of limited stretch. 2) Seeman's model is based on the prelytic leakage of K+ (97). The rate of water influx is decreased if a cell loses osmotically active substances; therefore, the degree of volume change is reduced at a given tonicity. The cell surface area remains constant, which implies that the membrane is not expandable. Furthermore, the reduction in osmotically active substances during the prelytic phase implies a Na—K exchange which is not equimolar. Therefore, a popula- tion of cells hemolyzes at a lower tonicity because the loss of osmotically active substances delays hemolysis. There appears to be evidence for and against both theories. Chan et al. have indicated that sucrose prevents hemolysis in red cells in a hypotonic medium by two mechanisms: 1) it counter- balances the colloid osmotic pressure and 2) it removes sufficient intracellular water to prevent critical membrane strain. Sucrose protected cells and unprotected residual cells, which have been returned to isotonicity, fail to reassume a biconcave disc shape. They suggest that this is due to a visco-elastic membrane which has undergone visco-plastic deformation (17). On the other hand, Jay and Rowlands have noticed there is a stress period of long duration during the hemolytic process in which the prelytic sphere shows a sudden reduction in volume. They reason that this is due to pre- lytic K+ leak (48), which supports Seeman's model. However, other investigators have demonstrated that the membrane is capable of stretching approximately 6-14% without subsequent lysis (15,100), whereas Jay and other investigators (33,49,57) have shown negligible increases in membrane extensibility when red blood cell membranes 21 are subjected to forces which result in surface area dilation. Fur- thermore, Rand and Burton have shown that the critical hemolytic volumes for fast and gradual hemolysis are the same (87). It is well known that the osmotic fragility of erythrocytes decreases as the temperature increases (2). Therefore, if membrane expansion is responsible for the decrease in fragility, then the critical hemolytic volume should increase with temperature (97). Murphy and others have reported that the isotonic volume and the critical hemolytic volume are not modified by temperature (72,97). Aloni et al. have indicated that membrane expansion involving the lipid layer is unlikely because liposomes from red blood cells show an increase in fragility with an increase in temperature (2). How- ever, Waugh and Evans have reported that the elasticity of the erythrocyte membrane increases as temperature increases (105) and conclude that a temperature modification of spectrin may change the solid elastic behavior of the membrane and thus increase its extensi- bility. Nevertheless, many investigators have reported prelytic K+ leak (24,80,83) and Poznansky and Solomon have shown a direct relation- ship between cell volume and K+ fluxes across the membrane. K+ influx is greater in shrunken cells; conversely, K+ efflux is greater in swollen cells (85). They conclude that the cell "uses" K+ fluxes as a mechanism to maintain is isotonic volume. Seeman has also demon- strated an increase in K+ leakage with an increase in temperature which could explain the decrease in osmotic fragility at higher temperatures (97). The explanation given for the prelytic K+ leak phenomenon is that pores can open up during the stress period when a cell is a prelytic sphere. When the pores become large enough to admit cations, the hydrated potassium ion, being smaller than the 22 hydrated sodium ion, is capable of leaking out of the membrane before any influx of sodium occurs (16). However, as demonstrated by Meryman and others, environmental factors such as the type of suspend- ing medium can greatly influence K+ leak as well as extensibility of the red cell membrane (69,111); therefore, caution should be applied when interpreting such results. Calcium - Effects on Red Blood Cell Function Introduction Erythrocytes are able to keep the calcium concentration in the cytosol several orders of magnitude below that in blood plasma. Several investigators have indicated that the intracellular concen- tration of free calcium is approximately 10—7 M per liter of red blood cells (92,96,103). Calcium, in minute quantities, is necessary for red blood cell metabolism; however, any disturbance of the steady- state distribution of calcium between medium and cells, for example an increase in intracellular calcium concentration, leads to physio- logically important effects in red blood cells. These disturbances are briefly discussed. Effects on Red Cell ATPases 2+ . . . . . . . Mg -Act1vated ATPase (ActomyOSin-like Protein) is activated in 2+ . the presence of Mg and ATP on the inner surface of the membrane. It has been shown recently that red blood cells retain their biconcave shape only when the activity of this enzyme remains unchanged (70). 2+ . . . When ATP and Mg are added to membrane preparations, this protein shows a change in viscosity. Like muscle actinomyosin, this protein + . is also inhibited by Cu2 and appears to be capable of contraction. 23 - . + Even though it appears that small concentrations of Ca2 are needed for its activation, it is thought that an increase in intracellular 2+ . 2+ . . . . . . Ca competes Wlth Mg for ATP, thus inhibiting its function. 2+ . . . . . . Ca -Activated ATPase is an enzyme which shows actiVity in the . . 2+ . . . . presence of millimolar Ca concentrations. This protein is thought to be identical to the major red blood cell protein known as . 2+ . . 2+ + "spectrin" (103). Mg prevents activation by Ca of the Ca2 - activated ATPase. Thus, increased intracellular calcium competes . 2+ . . . with Mg for ATP and pOSSibly regulatory Sites on spectrin. Further- . . 2+ . 2+ . more, it is thought that Mg -activated ATPase and Ca ~activated ATPase are expressions of the catalytic activity of spectrin (103). 2+ 2+ . . , (Ca + Mg )-Activated ATPase is the enzyme in the red blood . . . 2+ cell which regulates the concentration of intracellular Ca . It . 2+ . . . . 2+ requires Mg and is activated by micromolar concentrations of Ca 2+ . . . . . (95). Zn is thought to be an inhibitor of the protein (55). This enzyme appears to be the biochemical expression of the red blood cell . 2+ . membrane calCium pump (93,94). The low Ca intracellular concentra- . . . . 2+ tion is maintained by an ATP-dependent pump that moves Ca out through the membrane at a maximal rate, which exceeds the leak in the opposite . . 2+ . direction by a factor of 1000. Apparently, one mole of Ca is transported per mole of ATP hydrolyzed (59). The active form of the enzyme (holoenzyme) appears to be formed when a cytoplasmic activator called calmodulin combines with a less active form of the ATPase (apoenzyme). Calmodulin apparently acts as a modulator of the 2+ 2+ 2+ . . (Ca + Mg )-ATPase as well as regulator of free Ca by combining + . . with Ca2 first and then forming the holoenzyme complex With the ATPase protein (65). Furthermore, this modulator protein has been shown to activate cyclic nucleotide phosphodiesterase, adenylate 24 cyclase and, more weakly, regulates the MgZ+-ATPase (47). The phos- phorylation of the Ca2+-ATPase is a reversible process; the rate of dephosphorylation of the phosphoenzyme is increased by ADP (88). The low permeability of intact red blood cells to Ca2+ is not permanent but can be increased provided that the intracellular Ca2+ concentration is low (92). This transport protein is insensitive to ouabain (96). (Na+ + K+ + Mg2+)-Activated ATPase are a group of transport proteins which control the intracellular concentration of Na+ and K+. Based on in vitro studies, it is proposed that three sodium pumps exist in the red blood cell (46). Two are similar in that they require ATP and are inhibited by cardiac glycosides such as ouabain. They differ in that one requires K+ in the external medium, whereas the other requires Na+, the former being the more important for sodium and potassium exchanges (46). The third pump does not use ATP as an energy source; however, it does require glucose metabolism. It is not inhibited by ouabain, but is inhibited by ethacrynic acid. Mgz+ is required to activate the pumps (35) and it appears that two K+ ions are pumped in for every three Na+ ions pumped out. Cholesterol has an inhibitory effect on the protein by decreasing the fluidity of the lipid environment of the enzyme (19). Thus, a decrease in membrane cholesterol increases Na+ and K+ fluxes. Phospholipids have also been shown to be essential for enzyme integrity (89). It is thought that one effect of increased intracellular concentra- tions of Ca2+ is to block the action of the pump either directly by competing with Mg2+ for ATP or by binding to the membrane in such a manner as to affect the enzyme's environment (96). 25 Effects on Cation Fluxes It is known that under certain conditions calcium can promote the efflux of potassium. For example, the addition of fluoride to a cell suspension suppresses glycolysis and hence lowers the intracellu- lar ATP content. Consequently, active transport of K+ and Na+ ceases and net cation movement occurs. However, these exchanges are small in magnitude. If Ca2+ is added to this suspension, K+ efflux is 20 times faster than normal (76). The addition of the anti-glycolytic agent, iodoacetate (IAA), to a red blood cell suspension increases K+ efflux only in the presence of Ca2+ (Gérdos Phenomenon). Furthermore, there appears to be no concomitant influx of Na+ (60). Even though both compounds deplete the ATP content of the red blood cell, Ca2+ is still necessary to promote the efflux of K+. As noted earlier, Ca2+ can block the Na-K ATPase pump by competing with Mg2+ for ATP. However, this usually requires very high intracellular concentrations of the order of 100 uM per liter of red blood cells (96). The Gérdos phenomenon occurs with concentrations of intracellular Ca2+ as low as 3 x 10-7 M per liter of red blood cells (96). The Gérdos phenomenon has been investigated extensively and therefore will be briefly discussed. Apparently, K+ efflux occurs in four phases (60): 1) ATP depletion, a pre-condition for Ca2+ entry into the cells 2) The entry of calcium 3) The interaction between internal ionized calcium and the potassium gating process 4) The activation of the potassium channel. 26 It appears that ATP depletion is a prerequisite for increasing the cell membrane permeability to calcium (60,76). As noted earlier, glycolytic inhibitors which deplete the ATP content of red blood cells increase the cell's permeability to calcium by reducing the . . 2+ 2+ . acthity of the Ca + Mg -ATPase pump. Therefore, calCium permea- bility is under metabolic control. Furthermore, calcium uptake is strongly influenced by the monovalent cationic composition of the external medium (60) with permeability being increased in high concen- + . . . + . trations of Na and decreased in high concentrations of K . This has also been shown to occur in resealed red blood cell ghosts (54). Apparently, the ratio of ADP/ATP is also an important factor in determining calcium permeability (90). The site of the gating process + which eventually produces the efflux of K appears to be on the inside of the red cell membrane (4). Therefore, the concentration of internal 2+ . . Ca appears to be the controlling factor (96). The mechanism of . + . 2+ . . controlling K -gatlng by Ca 15 not well understood. It is thought 2+ + . that the Ca -dependent K channel may be activated by electron donors such as HS-glutathione, NADH or NADPH (38), which bring about a change in the redox state of some membrane components that makes intracellular 2+ . . . . . . + Ca more effective in eliCiting rapid K movements. Whatever the . . . . + . mechanism may be, it appears to be speCific for K because internal . + concentrations of Na do not change (96). . 2+ . . . Another system has been employed to increase Ca permeability in the red blood cell. The use of the ionophore A23187 (Calcimycin) 2+ . . . enhances Ca influx across the membrane by forming a complex Wlth + . . Ca2 which then traverses the plasma membrane. Therefore, it does not . . . 2+ . . . require ATP depletion to induce Ca permeability. The loading technique apparently does not alter the cell's ATP and 2,3-DPG 27 content or its morphology (91). However, this has not been confirmed as Plishker and Gitelman have shown that ATP concentrations can be radically reduced depending upon the concentration of ionophore and calcium in the external medium (77). Furthermore, they have observed marked ATP losses in the absence of Ca2+ accumulation, which implies a direct effect of the ionophore on the ATP hydrolysis system (29). Dunn has reported marked morphological changes when ATP-depleted systems are used to study cation effects on the red blood cell. Most of the cells appear to be sphero-echinocytes (29). A decrease in ATP of 5-15% of normal can cause these changes (64), which result from alterations in membrane skeletal structure. Effects on Membrane Structure and Function Approximately 95% of the calcium in the red blood cell is in the bound form (114), with 79% being bound to protein and 16% being bound to lipid. Over 80% of the lipid bound fraction is chelated to phospholipids (37), especially acidic phospholipids like phospho- tidylserine (61). The remaining portion of the calcium is located in the cytosol as ionized calcium and is regulated by the calcium pump (96). If intracellular calcium concentration exceeds 10-7 M per liter of red blood cells, then variouschangesoccur in the metabolism and function of human erythrocytes. Increased Ca2+ concentrations have been shown to cause red blood cell shrinkage with a concomitant increase in mean corpuscular hemoglobin concentration, an increase in the efflux of K+ accompanied by loss of water, a decrease in deforma- bility with stiffening of the membrane, and a severe change in morphology showing a disc to sphero-echinocyte transformation (31). Furthermore, a rapid hydrolysis of ATP occurs, possibly due to an 28 increased stimulation of the calcium pump or the binding of glycolytic chelators (ATP, 2,3,-Diphosphoglycerate) by ionized calcium. The efflux of K+ can occur with intracellular concentrations of calcium as low as 3 x lO-7 M per liter of red blood cells (96,114). The loss of osmotically active substances produces a dehydrated cell (xerocyte), thereby increasing the MCHC (64). These changes can be blocked if red blood cells are suspended in an isotonic solution of potassium chloride, which acts to inhibit passive loss of cellular K+ and, therefore, water (28,31). There are three explanations given for the molecular basis of decreased deformability (31,64). One pertains to the formation of "aggregates" caused by the direct interaction of Ca2+ with structural membrane components like spectrin and actin. Another explanation suggests that Ca2+ may in some manner contribute to the formation de novo of intermolecular disulfide bridges. The third explanation involves an enzyme called "transglutaminase." Lorand et al. have shown that this protein, which has a molecular weight of 80,000 (11), is normally latent in red blood cells. However, when intracellular concentrations of calcium increase to approximately 0.3 mM per liter of red blood cells (62), the enzyme is activated and causes the covalent fusion of membrane proteins by Y-glutamyl-E—lysine bridges, which produce polymers (63). The rate of the formation of the polymers is directly proportional to the intracellular concentration of Ca2+ (101). Anderson et al. have reported that the covalent linking involves spectrin and another structural protein (glycopeptide) other than actin (3). Allan and Mitchell have reported a different mechanism involving calcium (1). They have shown that an increase in intra- + . . . . . . . cellular Ca2 concentration stimulates enzymic actiVlties leading to 29 the formation of phosphatidic acid from membrane phospholipids. An intermediate, 1,2-diacylglycerol, is produced by a phosphorylating kinase reaction. This compound has been shown to be echinogenic (1). Other effects on the red blood cell membrane due to increased intracellular Ca2+ concentration include a decrease in hemolysis and osmotic fragility (58,66), a decrease in volume and permeability to solutes in resealed red blood cell ghosts (73,74), as well as facili- tating the resealing process (5,84). Increased calcium content in red blood cells has been impli- cated in some diseases. Sickle cells (drepanocytes) have been shown to have increased levels of calcium (14,30,75), possibly due to abnormal cation metabolism. Some observers have reported that red blood cells in hereditary spherocytosis show a decrease in Ca2+-ATPase activity (36), although this has not been confirmed (115). METHODS AND MATERIALS Introduction: Electrical Particle Sizing and Glutaraldehyde Fixation A red blood cell transversing a small orifice through which a known current is flowing gives rise to a countable resistive pulse, the magnitude of which is related to cell size (Coulter principle). Sizing a suspension of red blood cells results in a cumulative distribution or spectrum of such resistive pulses. A multi-channel analyzer can be used to receive and store these pulses according to their "electrical" size. The accumulation of pulses in each channel depends upon the size distribution in the red blood cell suspension. The spectrum of pulses is then recorded by printing out the number of pulses received in each channel, which is then used to determine the mean channel number. The mean channel number is determined by the following expression (10): Z cn Mean Channel Number (M.C.N.) = ( Zn ) - 0.5 where c is the channel number and n is the number of particles in each channel. The mean channel represents the channel into which all cells would fall if the total cell volume and number of cells were unchanged but all cells had the same volume. If the volume per channel is known, then multiplying this volume by the mean channel number will equal the mean cell volume (M.C.V.). However, electronic sizing of unfixed red blood cells causes several problems (67,113) which are 30 31 related to cell defonmability, medium composition, and flow rate. Most of these problems are overcome by the use of fixed cells (Figure 4). Nevertheless, the use of fixatives, such as glutaraldehyde, poses problems when used in high concentrations. Glutaraldehyde (penta- dialdehyde) fixes red blood cells by forming covalent amide bridges with lysinyl or 3-amino groups of valine in the intracellular hemo- globin, and in the membrane-associated proteins. Thus, it acts as a crosslinking agent. When high concentrations of glutaraldehyde are employed to fix red blood cell suspensions, the excess glutaraldehyde not only crosslinks proteins within a cell, but also crosslinks the surface proteins between cells, thus forming aggregates. Aggregate formation is enhanced when suspensions are centrifuged in order to remove the excess fixative. Fixing erythrocytes with glutaraldehyde in a hypotonic medium which is hemolytic poses further problems due to the presence of red blood cell ghosts and stromata which adhere to each other and also to the residual cells (71). Red blood cell aggregates invalidate sizing studies, where particle counts of single cells are desired. Therefore, the minimum amount of glutaraldehyde which produces complete fixation should be experimentally determined. This amount can vary depending upon the purity of glutaraldehyde and to what extent polymerization has occurred (71). Many investigators have reported that complete fixation of a red blood cell takes 10 to 20 minutes depending upon the suspending medium and the concentration of red blood cells (13,21,67,7l). Com- plete fixation has occurred if an erythrocyte is no longer osmotically active when placed in distilled deionized water (71). Mel and Yee have indicated that cell rigidification occurs within fifteen minutes using 0.05-0.10 percent glutaraldehyde (67) . Correy and Meiselman (21) 32 Figure 4. The effect of glutaraldehyde fixation on the sizing of red blood cells. The distribution curves of a popula— tion of unfixed red blood cells and fixed red blood cells using 0.125 percent glutaraldehyde are represented by figures A and C, respectively. The curve in figure A is bimodal, whereas the curve in figure C is unimodal and appears more Gaussian. Figure B shows an overlay of the two curves. Note the change in modality between the two curves. The modality of the distribution curve for fixed cells occurs at a higher channel number. The difference between the two curves in size distribution and modality is attributed to the differnce in deformability and flow-rate response of unfixed red blood cells versus fixed cells (67). 33 (A) 1 I (B) I 240 OBdb 05-- 0.4.- 0.2... 03-- 5:00 .33 cm: 45.9. mzp no 20.55: 02.. 0.4" 02-w- CHANNEL NUMBER FIGURE 34 have indicated that red blood cells suspended in a hypotonic medium which is lytic exhibit no hemolysis when glutaraldehyde uptake is greater than 70 micromoles per 1010 cells (approximately equal to one milliliter of packed red blood cells). Since many parameters affect red blood cell fixation and therefore particle sizing, it becomes apparent that the optimum concentration of glutaraldehyde employed should be experimentally determined. Determination of Appropriate Glutaraldehyde Concentration for Particle Sizing Blood was drawn by venipuncture from a normal volunteer, immedi- ately defibrinated, and used within three hours. The study was carried out at 25°C. To determine the appropriate glutaraldehyde concentration, 100 microliters of defibrinated blood was added to 5 milliliters of isotonic (300 mOsm/kg) sodium chloride containing either 0.05, 0.10, 0.50, 1.0, 2.0, 3.0 or 5.0 percent (v/v) gluta- raldehyde (reagent grade). These cell suspensions were fixed for either 0.5, 5.0, 10.0 or 30.0 minutes. All the tubes were run in triplicate. After the appropriate fixation time, 2 milliliters of the cell suspension were added to an equal proportion of distilled deionized water. The cells were immediately sized and the mean channel number was calculated for each pairing of concentration and fixation time. Comparisons of the mean channel numbers were made by using Student's t-test. The change in mean channel number due to swelling showed no significant difference when the minimum and maximum glutaraldehyde concentrations were compared with the minimum and maximum fixation times (Table 1). Therefore, the concentration of glutaraldehyde used throughout the remainder of this study was 0.125 percent (approximately equal to 2.5 millimoles of 35 Table 1. Comparison of mean channel number as a function of gluta- raldehyde concentration and fixation time at 300 mOsm Each line gives the average mean channel number for cells incubated at 25°C for 7 minutes in isosmolal sodium chloride solutions at stated glutaraldehyde concentrations (v/v) and fixation times. After fixation, cells were osmotically stressed by adding equal proportions of fixed cells and distilled deionized water and then sized using an electronic particle counter. Comparisons of the means are made for sequential pairs by standard statis- tical techniques (see text). Glutaraldehyde Fixation Mean Channel Con- concentration Time Number clu- in % (v/v) (minutes) Mean 1 S.D. t v p sion 0 05 0.5 43.8 i 0.2 1.000 4 0.1 0.45 N.S. 5 00 0.5 44 0 i 0.2 5 00 0.5 44.0 i 0.2 0.612 4 0.25uueucemeumeu e>uso Heposucs e e>um ou ceuusqeu me3 Emu 00mm um mpcooem ma Mom Guam HmCOHuuppe cm cecu .O eusmum cu meow uu we peumemme e>uso c0uuscuuumup ecu mu .ue>e30m .xuemmeoec me3 unassumm uecuHSM o: .o eusmww Cu meow ufi me ceueemmm e>uso :0uu5cuuumup ecu NH .ueue3 UeNHCOHeU peuuuumup cu pepcemmsmeu mez couusc HHeo ecu 6cm cepueomup mm3 uceumcuemsm ece .>D Hepoz Hemue>uc2 AumHv emsmuuuceu Hec0uuecueucH cm cu Emu oomm um mpcooem ma Mom peoswuuuceo meB mHHeo Hespumeu pee wumocm mo mc0flueasmom Uequ ecu maucueucoo Aueue3 beNACOHep peHHuumup :u maaeo ecu mcupcemmsmeu Use epwcepaeueusam ecu mau>05eu ueume penueucov COumcemmSm HHeo nexuw pepcemwsmeu denumuuo ecB .m3oHH0m mm peonmEe meB enquccoeu mcuccumm e .mumocm ecu e>OEeu oe .meupsum mauuum eueusooe Mow pepeec mu mHHeo Hespumeu mo meuum eaouuuem ecu xaco mcucueucoo e>uso GOAchuHumup HeUOEucs ecB .HepoEuc mu coucz .e>uso neuuscuuumup ecu zcexmee: SHHeuuuem :oHueasmom umocm ecu .euomeuece .< eusmum cu cmcu mmeH mu mHHeo Hespumeu mo necssc ecu ou peuemfioo we mumocm mo ueQEsc ecu .He>e30c “COuuesuum umHHEHm e Scum muusmeu m eusmuw cu e>uso ece .eESHo> Haeo uemueu m e>ec coucz .mHHeo Hespumeu mo COHueusmom ecu ou esp ucmuu ecu ou bezexm mu e>HDo ecB .mHHeo Hescuweu mo acuuscuuumup esuu ecu =pexmeE: :0umcemmsm Haeo ecu cu mumocm mo Mensa: emuea e .¢ eusmwm mo emeo ecu CH .eeue Max:3fii ecu >c ceucemeumeu mu mHHeo Hespumeu mo cOuueasmom ecu mo COuusc quumup esuu ecB .mx\EmOE omu BoHec meHuHHeHOEmo um mmeuum oquEmo ou neuoenQSm ceec mec coucz mHHeo pooHc pea mo c0uueasmom e MOM e>udo c0uu5cuuumuc ecu mcuucemeumeu we seem ceumo eue m use 4 meusmum CH peueuumsHHu me>uso ece .mumocm mau>OEeu How eswuccoeu aquacumm .m eusmum 4O ova cumin: Ana-5,310 O? L .9 a. l..N.O L. ed 4 0.0 .LNI'IOO 1133 038 'IVLOI 3H1 :IO NOILOVUd m 556.”. Ova Emmi-42 4w22<10 .0. .5 1.. 6.0 L... 6.0 L. Qo ““100 1133 038 'IVLOI 3H1 :IO NOIJLDVEH 41 setting of 2 with an amperage setting of 6.f The suspension in the counting vial was adjusted if the particle count was not between 15,000 and 25,000 counts per .25 milliliter. This particle count gave an adequate response time and number of pulses without introducing a large sizing error due to coincidence. The adjusted suspension was then sized using the Electrozone-Celloscope in conjunction with a Nuclear Data Multi-Channel Analyzer and ND-410 Display Monitorf until approximately 500,000 counts had accumulated. The numbers counted for each channel were then printed out on a teletype printer. The numbers were put into a Wang 700B calculator programmed with the expression given in the introduction on particle sizing. This program calculated the mean channel number for each red blood cell suspension that was sized. Duplicates for each osmolality were averaged and the mean was recorded. Mean channel numbers from several sizings at a given osmolality were then averaged. The average mean channel numbers at hypotonicity were compared to the average mean channel at isotonicity to obtain the mean fractional increase in volume. Swelling curves were constructed by plotting the mean fractional increase on the ordinate versus the corresponding corrected osmolality on the abscissa. The osmolalities had to be corrected due to the osmotic effect of the plasma and of the calcium chloride solutions, when used. Sizing experiments included runs with no calcium, 0.07, 2.20 and 4.40 millimoles of calcium per liter each having a different osmotic effect. Statistical analysis of the curves constructed from sizing experiments using various concentrations of calcium was then performed using Student's t-test. f . Particle Data, Inc., Elmhurst, IL. 42 The Effect of Cell Shape on Red Blood Cell Sizing As stated earlier, if the mean channel number (MC) and the volume per channel (V) are known, then the mean cell volume (MCV) can be calculated by the following expression: MCXV=MCV The volume per channel is also known as the calibration factor and will remain constant over a wide range of mean cell volumes providing that the cell shape during sizing has not altered the response of the instrument in such a way as to falsely increase or decrease the mean channel number. Therefore, if the mean cell volume and the mean channel number for a population of red blood cells at a given osmo- lality are knwon, then the calibration factor can be determined. The calibration factors can then be compared at different osmolalities, where shape changes occur, and analyzed by a regression line to determine whether or not the shape of the cell is affecting the response of the instrument. The mean channel number is obtained by sizing and the mean cell volume (MCV) can be calculated from the hematocrit (HCT) and the cell count, where HCT x 10 3_ Red blood cell count (millions/mm ) MCV= However, the packed cell volume used to determine the hematocrit is influenced by several factors, such as spinning time and packing factors associated with cell shape. For example, spheres do not pack as tightly as biconcave discs, which can readily deform; therefore, the amount of plasma trapped in the former is much greater than in the latter. Consequently, the hematocrit determined by the packed cell 43 volume will not be an accurate measurement of the proportional volume of the blood occupied by the erythrocytes. Therefore, an indirect method, using iron-59 labelled plasma, was employed to accurately assess the true hematocrit (7). The iron-59 hematocrit method is based on the principle that in a given volume of a red blood cell suspension, the red blood cells occupy a certain percentage of the volume and therefore the amount of radioactivity will be decreased compared to an equivalent volume of plasma. Therefore, an accurate measurement of the hematocrit can be achieved by a method which is not affected by cell shape. Blood was obtained by venipuncture from normal volunteers, immediately defibrinated, and centrifuged at 2500 rpm for 10 minutes in an International Centrifuge (IEC) Universal Model UV. The plasma was removed and saved for iron-59 labelling. The red blood cells were washed three times with 300 mOsm/kg sodium chloride, the pH of which was adjusted to 7.40 using a Beckman Model SS-2 pH meter. Five microcuries of iron—59 was added to 5 milliliters of plasma in a plastic tube with care being taken not to exceed the iron-binding capacity of the plasma. All tests were carried out at 25°C. The mean cell volume and mean channel number were determined as follows: all tests were performed in plastic tubes to prevent the adsorption of protein and radioactive iron onto the walls of the tubes. Red blood cells were osmotically stressed and fixed with a combina— tion of radioactive plasma and sodium chloride solutions at the following pre-lytic osmolalities (mOsm/kg): 300, 250, 230, 200 and 170. In order to obtain these osmolalities, adjustments had to be made because of the incorporation of plasma and "trapped saline" (from the washed red blood cells) into the sodium chloride solutions. 44 The osmolalities given above are the final osmolalities in the suspending medium. Therefore, the following stock solutions of sodium chloride, pH adjusted to 7.40 with NaOH, were used to make these adjustments: 300, 235, 210, 180, 150, 140, 120 and 75 (mOsm/kg). Two tubes labelled l and 2, each having a 10 milliliter capacity, were set up for each mean cell volume and mean channel number deter- mination, respectively. Tube number 1 contained 2 milliliters of iron-59 labelled plasma and 6 udlliliters cfi’ either 300, 180, 150, 120 or 75 (mOsm/kg) sodium chloride to represent the final osmolali- ties of 300, 250, 230, 200 and 170 (mOsm/kg), respectively. Tube number 2 contained 2 milliliters of iron-59 labelled plasma and 8 milliliters of either 300, 325, 210, 180 or 140 (mOsm/kg) sodium chloride to represent the final osmolalities of 300, 250, 230, 200 and 170 (mOsm/kg), respectively. At the start of the test, 4 milliliters of washed packed red blood cells were added to tube number 1 and osmotically stressed for 7 minutes; tube number 2 received 50 microliters of 25 percent glutaraldehyde (v/v). Both tubes were mixed gently. After mixing, an aliquot of the red blood cell suspension was removed and diluted with buffered isotonic saline using a Fisher Model 241 Automatic 9 This dilution was used to determine the cell count per Pipetter. .25 milliliter using the Celloscope-Electrozone particle counter. This cell count was corrected for coincidence and converted to millions per cubic millimeter. 9Fisher Scientific Co., Pittsburgh, PA. 45 After the 7 minute stress period, the red blood cell suspension was again well mixed; 2 milliliters were immediately transferred to a pre-weighed counting vial, re-weighed and then counted on a Nuclear Chicago gamma scintillation counterh for 5 minutes. The counts per minute were then determined and corrected for background. One hundred microliters of the suspension was added to tube number 2 containing the glutaraldehyde and fixed for 20 minutes. After the fixation period, the cell suspension was sized and the mean channel number was determined. The remaining portion of the suspension was used to fill a pre-weighed, 5 milliliter specific gravity bottle; the bottle was re-weighed and the specific gravity of the suspension was determined. After the counts per minute were recorded for the suspension, the counting vial and the specific gravity bottle con- taining the suspension were recombined with the suspension remaining in tube number 1 and centrifuged at 2500 rpm for 10 minutes. Two milliliters of the plasma were pipetted to another pre-weighed counting vial, re-weighed, and then counted for 5 minutes; the counts per minute were then determined and corrected for background. The remaining plasma was transferred to a second pre-weighed specific gravity bottle; the bottle was re-weighed, and the specific gravity of the plasma was determined. The specific gravity was used to calculate the volume from the weight of each sample. The counts per minute divided by the corrected volume gave the counts per minute per milliliter. The iron-59 * hematocrit (HCT) was determined by the following expression: hSearle and Co., Southfield, MI. 46 * Cp - Cs HCT = ° x 100 CP where Cp is the counts per minute per m1 of the plasma, Cs is the counts per minute per ml of the red blood cell suspension, and 100 is the factor to convert the volume fraction into volume percentage. The iron-59 hematocrit and the red blood cell count were then used to determine the mean cell volume (MCV) by the following expression: * HCT x 10 MCV = . . -_ RBC count (millions/mm3) The calibration factor was then calculated by dividing the mean cell volume by the mean channel number. Several calibration factors were determined at the osmolalities previously mentioned. A scatter diagram was constructed by plotting the calibration factor on the ordinate versus mean cell volume on the abscissa and regression analysis was subsequently performed using a Wang 7008 programmable calculator. Determination of the Cation Content of Osmotically Stressed Cells at Various Calcium Concentrations Blood was drawn by venipuncture from normal volunteers, immedi- ately defibrinated, and used within 3 hours. All tests were performed at 25°C. Stock sodium chloride solutions of 300, 160 and 120 mOsm/kg were used to provide an isotonic, a pre-lytic hypotonic, and a lytic hypotonic suspending medium. An isotonic solution of choline chloride (300 mOsm/kg) was employed as the solution to wash the red blood cells. Both the sodium chloride and choline chloride solutions were pH adjusted to 7.40 using a Beckman Model SS-2 pH meter. Calcium chloride solutions contained 114.5, 57.2 and 1.8 millimoles of calcium. These calcium concentrations were reduced to 4.40, 2.20 and 0.07 47 millimoles of calcium, respectively, when 200 microliters of the appropriate stock was added to 5 milliliters of isotonic or hypotonic chloride solution. Using a volumetric pipet, 5 milliliters of 300 mOsm/kg sodium chloride were pipetted into each of twelve, 7 milliliter plastic tubes, i.e., 3 tubes for each concentration of calcium used and 3 tubes containing no calcium. The same procedure was repeated for sodium chloride solutions containing 160 and 120 mOsm/kg. Two hundred microliters of the appropriate calcium chloride stock was added to those tubes receiving calcium. One hundred microliters of defibrinated whole blood was then added to each tube and osmotically stressed for 7 minutes. After the 7 minute period, the cells at 160 and 120 mOsm/kg were returned to isotonicity by adding to each tube 88 and 110 microliters, respectively, of a 4 M sodium chloride solution. All cell suspensions were then centrifuged at 1500 rpm for 10 minutes using an International Centrifuge (IEC) Universal Model UV. The supernatants were discarded and the cell buttons were washed four times with choline chloride. After discarding the fourth washing, the cells were resuspended in 0.7 milliliters choline chloride and the number of cells per .25 milliliter were determined using an Electrozone-Celloscope and a Fisher Automatic Dilutor containing buffered isotonic saline. All cell counts were coincidence corrected. The specimens were then kept in a freezer until further determinations were made. Freezing and thawing produced the hemolysates needed for the second phase of the experiment. The hemolysates were vortexed to insure proper mixing. To determine both the sodium and potassium values simultaneously, 0.5 milliliter of hemolysate and 50 microliters of 1500 milliequivalents 48 per liter lithium chloride solution were added to plastic 5 milliliter tubes containing 4.45 milliliters of distilled deionized water. The sodium and potassium in milliequivalents per liter of each diluted hemolysate were then read on an Instrumentation Laboratories Model 143 Flame Photometer.i The hemolysate was diluted 1:10, whereas the standard and the blank were commercially diluted 1:200. Therefore, each sodium and potassium value had to be corrected by dividing by 20. This gave the actual number of milliequivalents or millimoles per liter of the hemolysate. To find the number of millimoles of sodium or potassium per red blood cell, the following expression was devised to avoid correcting each value: M C x D x 4000 x 20 where M is the milliequivalents per liter for sodium or potassium read directly off the flame photometer, C is the coincidence corrected count per .25 milliliter read from the Celloscope, D is the dilution factor, 4000 converts .25 milliliter to liters, and 20 is the divisor for correcting the milliequivalents of sodium/potassium per liter due to differences in dilution between samples and standard. The effect of calcium on cation content was then analyzed using Student's t-test. i . . Instrumentation Laboratory, Inc., LeXington, MA. RESULTS The Effect of Red Cell Shape on Particle Sizing Linear regression analysis was performed using a Wang 700B pro- grammable calculator. The results indicate that red blood cell shape had almost no effect on particle sizing, r = -0.068 (Figure 6). Particle Sizing and Red Blood Cell Swelling The results indicate that electronic particle sizing with a modi- fied technique can be used for accurately determining the relative increase in red blood cell volume as well as calculating the critical hemolytic volume (Figure 7). Calcium Effects on the Relative Swelling of Red Blood Cells The effects of osmotically stressing red blood cells in solutions of sodium chloride containing 0.07, 2.20 and 4.40 millimoles of calcium are illustrated in Figure 8. Statistical analysis employing Student's t-test was performed at osmolalities that were: isotonic (300 mOsm/kg), hypotonic pre-lytic (160 mOsm/kg), and hypotonic lytic (120 mOsm/kg). The amount of swelling in sodium chloride solutions containing calcium was compared to the amount of swelling in sodium chloride solutions containing no calcium. The results are summarized in Tables 2, 3 and 4. Isosmolal and hyposmolal refer to isotonic and hypotonic, respectively. 49 50 .Heclcmec ecu >c ceucem .uceouem m.o mo emceHUCu ce .bov.m meB uouoeu COHueucuHmo ecu omH mo eEDHo> HHeo ceeE u .mcuuum eaouuuem e um “mmv.m mez uouoem COHueucuHeo ecu ow mo eEsHo> Haeo sees e ud neuQeu .mmo.ow uo :0uueu>e© puecceum m cuu3 gov.m mez :eeE eca .moo.ou u pce Hueo pooHc peu ecu mo emecm ecu cee3uec c0uueaeuuoo o: umOEHe mes euecu umcu peueoupcu mumxuece conmeumeu Heecuq .mcuuum eaofluuem co emecm eHoHuuem mo uoemme ecB .m eusmum 51 m 956.... 3...; ~13? duo 25: 3.. om. o... 2.: cm .63 :otu O O O O O O O O O O O iibl ii. :03 O O O o o O O .63 4 Ohm ('13NNVHO/ g u’) valor.) Mouyaanvo 52 Figure 7. The relative change in red blood cell volume using fixed red blood cells and an electronic particle counter. The figure illustrates the fractional increase in red blood cell volume, at 25°C, when 100 microliters of red blood cells were subjected to different concentrations of hypotonic sodium chloride containing no calcium. The cells were fixed with 0.125 percent glutaraldehyde and then sized. The curve follows the theoretical Ponder curves very closely and has a mean RW value of 46.2; the curves on either side, b and d, have RW values of 56.0 and 28.0, respectively. Thetmsh-barsrepresent the mean i one standard deviation. The numbers by each hash-bar represent the number of sizings determined at a given osmolality. The mean critical hemolytic volume (Vh) is the point where the maximum swelling occurs and shows a fractional increase in volume of 1.629. The curve also indicates: 1) that the red blood cells are not behaving as perfect osmometers and 2) that red blood cells are still present beyond the point of maximum swelling. These cells may not have swollen to their maximum or they may have become smaller due to excessive cation leak. ISOTONIC VOLUME FRACTION OF 1.8001? 1.700% 1.600-4 I I 1.500“ 1400+ I 1300‘ I 11004 I") 53 I0) (‘1) (4) (10) (8) (B) (7) (7) (8) 200 100 osmoumv (mOSM / kg) 66 d4 Tomcmr (1’) FIGURE 1 g... 8-4.. 01 0.1L 54 .eusuosuum msoceucEeEeuucu ecu oucu Eduoaeo mo coHueuomuoocu ecu ou esc eceucEeE ecu mo :oflueoumupumuu Ho >uuouumeae ecmucEeE cu emeeuoep e no xmea c0uumo e>ummeoxe ou esp ec See emeeuoec uceuemme ece .Esuoaeo mo meHOEuHHuE ov.v ocucueucoo mCOHuDHOm epuuoHco EducOm cuuz ceCuEueuep e>uso ecu mucemeumeu coucz .o eusmum mu coflumeoxe ecB .>uuHeHOEmo ue3oH e um usooo wecu uecu umeoxe .ueausum eue Ac>v meESHo> euu>a losec accuuuuo ceeE ece .eoceuwumeu oquEmo :u eweeHUCu cm mauuMUHUCH .meuuuaeaosmo uezou ecu mpuezou mumucw e>uso ecu .memeeuocu c0uueuuceocoo Eduoaeo ecu m4 .QOAumu>e© wheezeum eco u ceeE ecu enumep muecucmmc ecB .>He>uuoemmeu .0 use 0 .m meusmuw SQ peucemeumeu eue Eduoueo mo meHOEuHHuE ov.v 6cm om.m .no.o maucflmucoo chuuDHOm ecuuoHco ESApOm cu Auomm uev cecuaueuec me>uso mauHHeZm .meusmum uecuo ecu cu meuue>o an we ceucemeumeu Omae mu e>uuo ecB .Eduoaeo oc ocucueucoo ecouuDHOm ebuuoHco Enup0m Cu cuomm uev penueueuep e>uso mauauezm e we e>uueucemeumeu mu < eusmum .e>u:o ocuHHeBm coee mo COuuHom mou ecu meueuumsHHu >Hco eusmum ece .muueo pooHc new mo mcuaueze e>uueHeu ecu co Eduoueo mo muoemme ecB .m eusoum 55 0 $50.... 3. \ :33 550.80 8. 65 o: o! oo— .8 08.. . .OOCP 1.000— .005.— 3 \ Swag: 3350.60 00 09 09 0Q— 09 00— » .r 4 i 000— 1’ .60.: 1.80— a. 00 P 1 00w 3. \ .89.: E45280 0N— / As. \ smog: £350.60 0'— 00¢ \ 000... l .003 00 09 0a.. 03 :88 a(. P 52.. HR'IOA OINLOSI :IO ”MENU! 3Rn1OA OINOLOSI :IO NOILOVU! 56 Table 2. Comparisons of red cell relative swelling at 300 mOsm Each line gives the mean relative swelling as a fraction of the isosmolal volume for cells incubated at 25°C for 7 minutes in isosmolal sodium chloride solutions contain- ing stated concentrations of calcium. Cells were subse- quently fixed in 0.125% (v/v) glutaraldehyde for 20 minutes followed by centrifugation for 45 seconds at 2500 rpm. The supernatant was removed, the cells were resuspended in distilled deionized water and then sized using an electronic particle counter. Comparison of the means are made for sequential pairs by standard statistical techniques (see text). Fraction of Calcium Isosmolal Volume Concentration Mean i l S.D. t v 0 Conclusion 0.00 mM calcium 1.000 i 0.00 0.828 12 0.20.45 N.S. 0.07 mM calcium 1.392 t 0.016 0.00 mM calcium 1.391 t 0.022 2.108 19 0.01ec >Huceuemme mu Enuoueo .ue>e30c .cmx\EmOEV omH mo >uuHeHOEmo cm uc .oucouomxc ouuxaleum Ho eucouOmu eue couc3 meuuuHeHOEmo ue uceucoo Enummeuom Ho EduCOm cu emceco Heuuceuwcsm 0c mu euecu uecu meueOupcu eusmuu ecE .uceucoo COHueo uceae>ocoe Heuou ecu How c0uueu>e© pueoceum eco u ceeE ecu eueoupcw mueclcmec ecB .meHOEuHHuE Ho muceue>usqeuuaufi cu uceucoo COuueo uceue>ocoe Heuou ecu mucemeumeu uec euuuce ecu Use .ESHGOm mo meHOEuHHuE uo muceae>usqeuuaue wucemeuaeu uec ecu mo c0uuuom euucz ecu .Esuwmeuom mo meHOEuHHuE uo muceue>usveuHHuE mucemeumeu uec ecu mo c0uuuom pepecm eca .>He>uuoeameu .0 use 0 .m mueuuea ecu >c peuocep eue EduoHeo mo meHOEuHHuE ov.v pee om.m .bo.o mCHCueucoo mCOHuDHOm epuuoHco Enupom cu cecusueuep meupsum COuueO .4 ueuueu ecu >c peuocec eue Eduoueo o: mCHCueucoo mCOuusHOm epuuoHco EDHGOm nu pecuE lueuep meupsum :oHueO .uceucoo c0uueo Hueo cooHc peu co Eduoaeo mo uoemme ecB .m eusmum 61 0 5.50....— 3: \ 28.5 >tu<40¢¢00 A9 80 A9 A5 A3 A9 A3 A5 OOOOOO O O O OOIOOO O OOO OOOO O OOOOOO O O O O O O O O O I O O OOOOI O O OOOO O OOOOOO O O... O 2: I o O I O I O u a a 00- .. 000-0 000-00 00.... .0000. 0 o O o u I o 0 O o u n o u a o o a a u o a a n a 0 0 O O O O O O O O O I 0 O O l l I I I O O O O O O O O O con... to... one-no .00... on... use... O O O I one... ”one”. one... I O O O O O O O I one... on... coo-00 o 0 0 a o n 0 o 0 000... o I o o 0 a m .0. a. 2: 1 I00w ct0L x (00: mm) 1N31NOO Home 62 Table 5. Comparisons of red cell sodium content at 300 mOsm Each line gives the mean sodium content in mM x 1013 per cell, for cells incubated at 25°C for 7 minutes, in isosmolal sodium chloride solutions containing stated concentrations of calcium. Comparisons of the means are made for sequential pairs by standard statis- tical techniques (see text). Red Cell Calcium Sodium Concentration Mean i 1 S.D. t v 0 Conclusion 0.00 mM calcium 9.6 i 0.9 0.495 14 0.300.45 N.S. 2.20 mM calcium 9.8 i 0.8 0.07 mM calcium 9.8 i 0.5 0.703 14 0.200.45 N.S. 2.20 mM calcium 89.6 i 4.1 0.00 mM calcium 89.4 i 2.9 0.000 14 p>0.45 N.S. 4.40 mM calcium 89.4 i 1.6 0.07 mM calcium 89.0 i 1.6 0.366 14 0.30.45 N.S. 4.40 mM calcium 89.4 i 1.6 64 Table 7. Comparisons of red cell total cation content at 300 mOsm Each line gives the mean total cation content (sodium and potassium) in mM x 1013 per cell, for cells incubated at 25°C for 7 minutes, in isosmolal sodium chloride solutions containing stated concentrations of calcium. Comparisons of the means are made for sequential pairs by standard statistical techniques (see text). Total Cation Calcium Content Concentration Mean i 1 S.D. t v 0 Conclusion 0.00 mM calcium 99.1 f 2.6 0.258 14 p=0.4 N.S. 0.07 mM calcium 98.8 i 1.7 0.00 mM calcium 99.1 i 2.6 0.113 14 p>0.45 N.S. 2.20 mM calcium 99.3 i 2.0 0.00 mM calcium 99.1 i 2.6 0.244 14 0.40.45 N.S. 4.40 mM calcium 99.4 i 2.0 Tabl 65 e 8. Comparisons of red cell sodium content at 160 mOsm . . . . 13 Each line gives the mean sodium content in mM x 10 per cell, for cells incubated at 25°C for 7 minutes, in hyposmolal sodium chloride solutions containing stated concentrations of calcium. Comparisons of the means are made for sequential pairs by standard statistical techniques (see text). Red Cell Calcium Sodium Concentration Mean i l S.D. t v 0 Conclusion 0.00 mM calcium 9.5 i 0.5 1.721 14 0.050.45 N.S. 2.20 mM calcium 9.5 i 0.7 0.00 mM calcium 9.5 i 0.5 0.299 14 0.300.45 N.S. 2.20 mM calcium 89.3 i 2.0 0.07 mM calcium 89.2 i 1.7 0.000 14 p>0.45 N.S. 4.40 mM calcium 89.2 i 2.1 2.20 mM calcium 89.3 i 2.0 0.091 14 p>0.45 N.S. 4.40 mM calcium 89.2 i 2.1 67 Table 10. Comparisons of red cell total cation content at 160 mOsm Each line gives the mean total cation content (sodium and potassium) in mM x 1013 per cell, for cells incubated at 25°C for 7 minutes, in hyposmolal sodium chloride solu- tions containing stated concentrations of calcium. Com- parisons of the means are made for sequential pairs by standard statistical techniques (see text). Total Cation Calcium Content Concentration Mean i l S.D. t v p Conclusion 0.00 mM calcium 99.7 i 1.9 0.761 14 0.20.45 N.S. 4.40 mM calcium 98.8 i 2.1 68 Table 11. Comparisons of red cell sodium content at 120 mOsm Each line gives the mean sodium content in mM x 1013 per cell, for cells incubated at 25°C for 7 minutes, in hyposmolal sodium chloride solutions containing stated concentrations of calcium. Comparisons of the means are made for sequential pairs by standard statistical techniques (see text). Red Cell Calcium Sodium Concentration Mean i 1 S.D. t v 0 Conclusion 0.00 mM calcium 24.7 i 1.4 0.219 14 0.4